1. Field of the Invention
The present invention relates to detection techniques in bio-analysis, particularly detection of bio-separation through a separation channel, and more particularly to optics relating to application of incident radiation at and detection of output radiation from a detection zone along the separation column, e.g., a capillary column. The present invention further relates to a bio-separation instrument incorporating the detection technique, in particular a capillary electrophoresis instrument.
2. Description of Related Art
Currently, most of bio-separation tools applied in the laboratories utilize slab gel based electrophoresis technologies, which have routinely been used for bio-analysis of bio-molecules (i.e. DNA, Protein & Carbohydrate) applications since their inception more than 20 years ago. However, slab gel electrophoresis for bio-analysis is labor intensive and needs to be drastically improved in terms of resolving power, throughput and cost per sample.
Capillary electrophoresis (CE) is a micro fluidic approach to gel-electrophoresis (micro-channel device to simplify gel-electrophoresis), whose greatest advantage is its diverse range of applications. CE technology is commonly accepted by the biotechnology industry specifically in the nucleic acid-based testing as a reliable, high resolution and highly sensitive detection tool, and CE has been applied for protein, carbohydrate and DNA-related analyses such as oligonucleotides analysis, DNA sequencing, and dsDNA fragments analysis. CE is commonly avoided in routine analysis because it is reputed to be a troublesome technique with high failure rates. However this is no longer true because instrument manufacturers have drastically improved instrument design and overall CE knowledge has increased. There are three key factors for reducing failure rate and producing accurate, precise and robust CE data: operator training, system stability, and operation ease of the instrument with low maintenance.
Capillary Electrophoresis Immunoassay Analysis (CEIA) has recently emerged as a new analytical technique, when combined with sensitive detection methods such as Laser Induced Fluorescence (LIF), offers several advantages over the conventional immunoassays. CEIA can perform rapid separations with high mass sensitivity, simultaneously determine multiple analytes and is compatible with automation. Use of CE and florescence labeled peptides can be used to detect abnormal prion protein in the blood of animals. One such CE-based noncompetitive immunoassay for Prion Protein using Fluorescein isithiocyanate (FITC)-labeled Protein A as Fluorescent probe method has successfully been applied for testing blood samples from scrapie-infected sheep.
Further, immunoassays are commonly used in biotechnology for the detection and quantification of host cell contaminants. The free-solution approach by CE with fluorescence type detection has brought an exciting alternative to solid-phase immunoassay. The CE with fluorescent type detection eliminates antigen immobilization and avoids many solid-phase-associated problems. This methodology makes use of either a purified antigen labeled with stable fluorescent dye (i.e. FITC) or an affinity probe labeled with the dye (direct assay).
Without a doubt, CE with laser-induced fluorescence (LIF) is one of the most powerful analytical tools for rapid, high sensitivity and high-resolution dsDNA analysis and immunoassay analysis applications. However, the current selling price for CE-based LIF systems is much more expensive than traditional slab-gel based bio-analysis systems due to the complicated optical detection mechanism. The expensive CE-based systems are thus out of reach for all but a few well-funded laboratories and seems to be a high-cost barrier for the expansion of immunoassay type analysis applications/business.
There is a need for a system with a less complex optical detection mechanism to reduce costs, which complements simplicity in operation, rapid analysis with high efficiency, sensitivity and throughput.